Neutrophil mobilization to inflammatory sites is a multi-step process which involves the reversible adhesion to specific endothelial plasma membrane molecules, upregulation of selected neutrophil adhesion molecules during chemoattractant exposure, and migration (chemotaxis) through endothelial gaps and the basement membrane into tissues. The present study was undertaken to examine the changes that occur in adhesion molecule expression during neutrophil chemotaxis in vitro. Two populations of neutrophils are easily identified and separated with the in vitro polycarbonate membrane system; one population does not migrate to chemoattractant (nonmigrating population) and remains on the upper surface of the membrane, whereas the other population migrates through the membrane pores to the lower surface of the membrane (migrating population). Neutrophils, incubated in suspension with or without the N-formyl peptide (FMLP), were compared with the migrating and nonmigrating subpopulations which were exposed to a gradient of FMLP. Neutrophils were stained with a panel of adhesion molecule monoclonal antibodies which recognize: the leukocyte-cell adhesion molecule family (Leu-CAM), CD11a, CD11b, and CD11c; the platelet/ endothelial cell adhesion molecule-1 (PECAM-1), CD31; leukosialin, CD43; and, the homing receptor Pgp-1, CD44. Flow cytometric analysis of the panel revealed that the migrating subpopulation consistently exhibited less surface expression of these adhesion molecules than the nonmigrating subpopulation. CD44, CD43, and CD11a were the most dramatically decreased (49-60%), whereas, CD11c, CD31, and CD11b were moderately decreased (25-38%). Expression of a control antigen, CD15, remained unchanged in migrating and nonmigrating neutrophils. These results indicate that neutrophil adhesion molecule expression is differentially downregulated during neutrophil chemotaxis. This experimental system provides a means for quantifying the expression of myeloid antigens which are important in inflammatory responses.